Isolation and purification of the putative natriuretic hormone (NH) will continue, using high performance liquid chromatography (HPLC). Partial purification of two factors has been achieved. One is a low molecular weight inhibitor of active sodium transport in toad bladder antinatriferic factor, AF). The other is a higher molecular weight (HMW) natriuretic factor with no antinatriferic activity. Preliminary studies suggest that trypsin digestion of HMW factor produces the AF, suggesting that HMW factor is a precursor of AF. Additonal studies to confirm this observation will be performed. Preliminary studies indicate that AF is an inhibitor of NaK ATPase in vitro. Additional studies are planned using a commercially available purified brain NaK ATPase. Preliminary studies show that HPLC extracts which contain both factors described above also contain a substance (or substances) which is recognized by anti-digoxin antibodies. This suggests that these endogenous digoxins (called "endoxins") may be the natriuretic hormone and its precursor. Further studies are under way to test this hypothesis. The possibility that NH and/or its precursor might be a vasoconstrictor which plays a role in the pathophysiology of hypertension is being investigated. The response of arterioles to injected norepinephrine (NE) is observed directly in a cremaster muscle preparation before and after injections of NH preparation. Preliminary studies have demonstrated that the HPLC fraction containing the HMW factor from volume-expanded dogs causes increased sensitivity of arterioles to exogenous NE. These studies are continuing.